Epithelial GPR35 protects from Citrobacter rodentium infection by preserving goblet cells and mucosal barrier integrity

Goblet cells are essential for maintaining intestinal health and for the defense against invasive bacterial infection. However, the molecular pathways that regulate goblet cell function remain largely unknown. Although GPR35 is highly expressed in colonic epithelial cells, its importance in promoting the epithelial barrier is unclear. Here we found that epithelial Gpr35 plays a critical role in goblet cell function. Genetic deletion of Gpr35 in epithelial cells but not in from macrophages results in goblet cell depletion and dysbiosis, rendering these mice more susceptible to Citrobacter rodentium infection. Mechanistically, scRNA-seq analysis indicates that signaling of epithelial Gpr35 is essential to maintain normal pyroptosis levels in goblet cells. Our work shows how the epithelial presence of Gpr35 is a critical element for the function of goblet cell-mediated symbiosis between host and microbiota.

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Entamoeba histolytica-Induced Mucin Exocytosis Is Mediated by VAMP8 and Is Critical in Mucosal Innate Host Defense

mBio ◽

10.1128/mbio.01323-17 ◽

2017 ◽

Vol 8 (5) ◽

Cited By ~ 14

Author(s):

Steve Cornick ◽

France Moreau ◽

Herbert Y. Gaisano ◽

Kris Chadee

Keyword(s):

Epithelial Cells ◽

Entamoeba Histolytica ◽

Host Defense ◽

Critical Role ◽

Single Layer ◽

Goblet Cells ◽

Mucus Secretion ◽

Mucosal Barrier ◽

Proinflammatory Response

ABSTRACT Intestinal mucus secretion is critical in maintaining mucosal host defense against a myriad of pathogens by preventing direct association with the epithelium. Entamoeba histolytica specifically binds colonic MUC2 mucin and also induces potent hypersecretion from goblet cells; however, characterization of the nature of the mechanisms controlling mucus release remains elusive. In this report, we identify vesicle SNARE vesicle-associated membrane protein 8 (VAMP8) present on mucin granules as orchestrating regulated exocytosis in human goblet cells in response to the presence of E. histolytica. VAMP8 was specifically activated during E. histolytica infection, and ablation of VAMP8 led to impaired mucin secretion. As a consequence, loss of VAMP8 increased E. histolytica adherence to epithelial cells associated with enhanced cell death through apoptosis characterized by caspase 3 and 9 cleavages and DNA fragmentation. With the mucosal barrier compromised in Vamp8 −/− animals, E. histolytica induced an aggressive proinflammatory response with elevated levels of interleukin-1 alpha (IL-1α), IL-1β, and tumor necrosis factor alpha (TNF-α) secretion. This report is the first to characterize regulated mucin exocytosis in intestinal goblet cells in response to a pathogen and the downstream consequences of improper mucin secretion in mucosal barrier defense. IMPORTANCE The intestinal tract is exposed to countless substances and pathogens, and yet homeostasis is maintained, in part by the mucus layer that houses the microbiota and spatially separates potential threats from the underlying single layer of epithelium. Despite the critical role of mucus in innate host defense, characterization of the mechanisms by which mucus is secreted from specialized goblet cells in the gut remains elusive. Here, we describe the machinery that regulates mucus secretion as well as the consequence during infection with the colonic pathogen Entamoeba histolytica. Abolishment of the key machinery protein VAMP8 abrogated mucus release in cultured human colonic goblet cells and during E. histolytica infection in Vamp8 −/− mice, which showed enhanced amoeba contact and killing of epithelial cells, triggering a potent proinflammatory response. This report highlights the importance of the VAMP8 secretory machinery in facilitating mucus release from intestinal goblet cells and the dire consequences that occur during disease pathogenesis if these pathways are not functional. IMPORTANCE The intestinal tract is exposed to countless substances and pathogens, and yet homeostasis is maintained, in part by the mucus layer that houses the microbiota and spatially separates potential threats from the underlying single layer of epithelium. Despite the critical role of mucus in innate host defense, characterization of the mechanisms by which mucus is secreted from specialized goblet cells in the gut remains elusive. Here, we describe the machinery that regulates mucus secretion as well as the consequence during infection with the colonic pathogen Entamoeba histolytica. Abolishment of the key machinery protein VAMP8 abrogated mucus release in cultured human colonic goblet cells and during E. histolytica infection in Vamp8 −/− mice, which showed enhanced amoeba contact and killing of epithelial cells, triggering a potent proinflammatory response. This report highlights the importance of the VAMP8 secretory machinery in facilitating mucus release from intestinal goblet cells and the dire consequences that occur during disease pathogenesis if these pathways are not functional.

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Modulation of Conjunctival Goblet Cell Function by Inflammatory Cytokines

Mediators of Inflammation ◽

10.1155/2013/636812 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 44

Author(s):

L. Contreras-Ruiz ◽

A. Ghosh-Mitra ◽

M. A. Shatos ◽

D. A. Dartt ◽

S. Masli

Keyword(s):

Sjögren’S Syndrome ◽

Inflammatory Cytokines ◽

Goblet Cell ◽

Ocular Surface ◽

Cell Function ◽

Sjögren's Syndrome ◽

Goblet Cells ◽

Sjogren’S Syndrome ◽

Sjogren's Syndrome ◽

Sjögren‘S Syndrome

Ocular surface inflammation associated with Sjögren’s syndrome is characterized by a loss of secretory function and alteration in numbers of mucin secreting goblet cells. Such changes are a prominent feature of ocular surface inflammatory diseases and are attributed to inflammation; however, the exact effect of the inflammatory cytokines on conjunctival goblet cell function remains largely unknown. In this study, we developed a primary culture of mouse goblet cells from conjunctival tissue and evaluated the effects on their function by inflammatory cytokines detected in the conjunctiva of mouse model of Sjögren’s syndrome (Thrombospondin-1 deficient mice). We found that apoptosis of goblet cells was primarily induced by TNF-αand IFN-γ. These two cytokines also inhibited mucin secretion by goblet cells in response to cholinergic stimulation, whereas IL-6 enhanced such secretion. No changes in secretory response were detected in the presence of IL-13 or IL-17. Goblet cells proliferated to varying degrees in response to all the tested cytokines with the greatest response to IL-13 followed by IL-6. Our results therefore reveal that inflammatory cytokines expressed in the conjunctiva during an ocular surface disease directly disrupt conjunctival goblet cell functions, compromising the protective function of tears, thereby contributing to ocular surface damage.

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Bifidobacterium dentiumFortifies the Intestinal Mucus Layer via Autophagy and Calcium Signaling Pathways

mBio ◽

10.1128/mbio.01087-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 15

Author(s):

Melinda A. Engevik ◽

Berkley Luk ◽

Alexandra L. Chang-Graham ◽

Anne Hall ◽

Beatrice Herrmann ◽

...

Keyword(s):

Calcium Signaling ◽

Signaling Pathways ◽

Goblet Cell ◽

Cell Function ◽

Goblet Cells ◽

Mucus Layer ◽

Content Type ◽

Intestinal Mucus ◽

Intestinal Mucus Layer

ABSTRACTMuch remains unknown about how the intestinal microbiome interfaces with the protective intestinal mucus layer.Bifidobacteriumspecies colonize the intestinal mucus layer and can modulate mucus production by goblet cells. However, selectBifidobacteriumstrains can also degrade protective glycans on mucin proteins. We hypothesized that the human-derived speciesBifidobacterium dentiumwould increase intestinal mucus synthesis and expulsion, without extensive degradation of mucin glycans.In silicodata revealed thatB. dentiumlacked the enzymes necessary to extensively degrade mucin glycans. This finding was confirmed by demonstrating thatB. dentiumcould not use naive mucin glycans as primary carbon sourcesin vitro. To examineB. dentiummucus modulationin vivo, Swiss Webster germfree mice were monoassociated with live or heat-killedB. dentium. LiveB. dentium-monoassociated mice exhibited increased colonic expression of goblet cell markersKrüppel-like factor 4(Klf4),Trefoil factor 3(Tff3),Relm-β,Muc2, and several glycosyltransferases compared to both heat-killedB. dentiumand germfree counterparts. Likewise, liveB. dentium-monoassociated colon had increased acidic mucin-filled goblet cells, as denoted by Periodic Acid-Schiff-Alcian Blue (PAS-AB) staining and MUC2 immunostaining.In vitro,B. dentium-secreted products, including acetate, were able to increase MUC2 levels in T84 cells. We also identified thatB. dentium-secreted products, such as γ-aminobutyric acid (GABA), stimulated autophagy-mediated calcium signaling and MUC2 release. This work illustrates thatB. dentiumis capable of enhancing the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways, with a net increase in mucin production.IMPORTANCEMicrobe-host interactions in the intestine occur along the mucus-covered epithelium. In the gastrointestinal tract, mucus is composed of glycan-covered proteins, or mucins, which are secreted by goblet cells to form a protective gel-like structure above the epithelium. Low levels of mucin or alterations in mucin glycans are associated with inflammation and colitis in mice and humans. Although current literature links microbes to the modulation of goblet cells and mucins, the molecular pathways involved are not yet fully understood. Using a combination of gnotobiotic mice and mucus-secreting cell lines, we have identified a human-derived microbe,Bifidobacterium dentium, which adheres to intestinal mucus and secretes metabolites that upregulate the major mucin MUC2 and modulate goblet cell function. Unlike otherBifidobacteriumspecies,B. dentiumdoes not extensively degrade mucin glycans and cannot grow on mucin alone. This work points to the potential of usingB. dentiumand similar mucin-friendly microbes as therapeutic agents for intestinal disorders with disruptions in the mucus barrier.

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Studies on the Digestion of Wool by Insects V. The Goblet Cells in the Midgut of Larvae of the Clothes Moth (Tineola Bissellielal (Humm.)) And Other Lepidoptera

Australian Journal of Biological Sciences ◽

10.1071/bi9520169 ◽

1952 ◽

Vol 5 (1) ◽

pp. 169 ◽

Cited By ~ 2

Author(s):

DF Waterhouse

Keyword(s):

Epithelial Cells ◽

Goblet Cell ◽

Staining Technique ◽

Basic Structure ◽

Goblet Cells ◽

Internal Cavity ◽

Frequency Of Occurrence ◽

Regenerative Cells ◽

Pass Through ◽

Columnar Cells

Goblet cells and columnar cells occur, together with regenerative cells, in the midgut epithelium of lepidopterous larvae. The columnar cells have an appearance typical of the simple epithelial cells that occur in the midgut of many insects. The goblet cells are highly differentiated and, although there are marked variations between species, such as in frequency of occurrence, in shape, in staining reactions, and so on, their basic structure is very similar. Bodian's 'ProtargoI' staining technique provides excellent differentiation of goblet cells. Each goblet cell has a basally situated nucleus and contains an internal cavity, which is bordered by a faintly striated lining. No opening permitting direct movement of material from the cavity into the lumen has been observed. Available evidence suggests that materials moving out of the cavity pass through a bounding membrane. Unlike columnar cells, goblet cells do not possess a striated border on their lumen surface.

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The impact of oligosaccharide content, glycosidic linkages and lactose content of galacto-oligosaccharides (GOS) on the expression of mucus-related genes in goblet cells

Food & Function ◽

10.1039/d0fo00064g ◽

2020 ◽

Vol 11 (4) ◽

pp. 3506-3515 ◽

Cited By ~ 2

Author(s):

Susana Figueroa-Lozano ◽

Chengcheng Ren ◽

Huifang Yin ◽

Hien Pham ◽

Sander van Leeuwen ◽

...

Keyword(s):

Molecular Structure ◽

Direct Effect ◽

Goblet Cell ◽

Cell Function ◽

Goblet Cells ◽

The Impact

Galacto-oligosaccharides (GOS) are molecules known to impact goblet cell function and mucus composition. Our results show that their molecular structure have a direct effect on goblet cells and impact mucus synthesis-related genes.

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Type III interferon signaling restricts enterovirus 71 infection of goblet cells

Science Advances ◽

10.1126/sciadv.aau4255 ◽

2019 ◽

Vol 5 (3) ◽

pp. eaau4255 ◽

Cited By ~ 27

Author(s):

Charles Good ◽

Alexandra I. Wells ◽

Carolyn B. Coyne

Keyword(s):

Goblet Cell ◽

Intestinal Epithelium ◽

Cell Function ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Goblet Cells ◽

Neurological Complications ◽

Ev71 Infection ◽

Apical Surface ◽

Type Iii

Recent worldwide outbreaks of enterovirus 71 (EV71) have caused major epidemics of hand, foot, and mouth disease with severe neurological complications, including acute flaccid paralysis. EV71 is transmitted by the enteral route, but little is known about the mechanisms it uses to cross the human gastrointestinal tract. Using primary human intestinal epithelial monolayers, we show that EV71 infects the epithelium from the apical surface, where it preferentially infects goblet cells. We found that EV71 infection did not alter epithelial barrier function but did reduce the expression of goblet cell–derived mucins, suggesting that it alters goblet cell function. We also show that the intestinal epithelium responds to EV71 infection through the selective induction of type III interferons (IFNs), which restrict EV71 replication. Collectively, these findings define the early events associated with EV71 infections of the human intestinal epithelium and show that host IFN signaling controls replication in an IFN-specific manner.

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Phosphodiesterase expression in human epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.275.4.l694 ◽

1998 ◽

Vol 275 (4) ◽

pp. L694-L700 ◽

Cited By ~ 19

Author(s):

Lyndon C. Wright ◽

Joachim Seybold ◽

Annette Robichaud ◽

Ian M. Adcock ◽

Peter J. Barnes

Keyword(s):

Epithelial Cells ◽

Cell Function ◽

Critical Role ◽

A549 Cells ◽

Airway Epithelial Cells ◽

Epithelial Cell Line ◽

Airway Epithelial ◽

Bioactive Lipids ◽

Rt Pcr ◽

Inflammatory Conditions

Epithelial cells play a critical role in airway inflammation and have the capacity to produce many inflammatory mediators, including bioactive lipids and proinflammatory cytokines. Intracellular levels of cAMP and cGMP are important in the control of inflammatory cell function. These cyclic nucleotides are inactivated via a family of phosphodiesterase (PDE) enzymes, providing a possible site for drug intervention in chronic inflammatory conditions. We studied the expression of PDE activity in an epithelial cell line (A549) and in primary human airway epithelial cells (HAECs). We measured PDE function using specific inhibitors to identify the PDE families present and used RT-PCR to elucidate the expression of PDE isogenes. Both A549 cells and HAECs predominantly expressed PDE4 activity, with lesser PDE1, PDE3, and PDE5 activity. RT-PCR identified HSPDE4A5 and HSPDE4D3 together with HSPDE7. Inhibition of PDE4 and PDE3 reduced secretion by these cells. Epithelial PDE may be an important target for PDE4 inhibitors in the development of the control of asthmatic inflammation, particularly when delivered via the inhaled route.

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Impact of P2X7 Purinoceptors on Goblet Cell Function: Implications for Dry Eye

International Journal of Molecular Sciences ◽

10.3390/ijms22136935 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6935

Author(s):

Donald G. Puro

Keyword(s):

Dry Eye ◽

Goblet Cell ◽

Cell Function ◽

Tear Film ◽

Goblet Cells ◽

High Gain ◽

High Temporal Resolution ◽

Triggered Release ◽

Voltage Gated Calcium Channels ◽

Conjunctival Goblet Cells

By providing ~70% of the eye’s refractive power, the preocular tear film is essential for optimal vision. However, its integrity is often jeopardized by environmental and pathologic conditions that accelerate evaporation and cause sight-impairing dry eye. A key adaptive response to evaporation-induced tear film hyperosmolarity is the reflex-triggered release of tear-stabilizing mucin from conjunctival goblet cells. Here, we review progress in elucidating the roles of ion channels in mediating this important exocytotic response. Much is now known about the modulatory impact of ATP-sensitive potassium channels, nonspecific cation channels and voltage-gated calcium channels. Recently, we discovered that during unremitting extracellular hyperosmolarity, P2X7 receptor/channels also become activated and markedly impair goblet cell viability. However, our understanding of possible adaptive benefits of this P2X7 activation remains limited. In the present study, we utilized high-temporal resolution membrane capacitance measurements to monitor the exocytotic activity of single goblet cells located in freshly excised rat conjunctiva. We now report that activation of P2X7 purinoceptors boosts neural-evoked exocytosis and accelerates replenishment of mucin-filled granules after exocytotic depletion. Thus, P2X7 activation exerts a yin-yang effect on conjunctival goblet cells: the high-gain benefit of enhancing the supply of tear-stabilizing mucin is implemented at the high-risk of endangering goblet cell survival.

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Intestinal Goblet Cells Play a Protective Role Against GVHD Via a Lypd8-Dependent Manner after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2018-99-115510 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 64-64

Author(s):

Takahide Ara ◽

Daigo Hashimoto ◽

Eiko Hayase ◽

Noizat Clara ◽

Ryu Okumura ◽

...

Keyword(s):

Epithelial Cells ◽

Quantitative Pcr ◽

Goblet Cell ◽

Bacterial Translocation ◽

Colonic Mucosa ◽

Bacterial Load ◽

Goblet Cells ◽

Protective Role ◽

Dependent Manner ◽

Hematopoietic Stem

Abstract [Introduction] Emerging evidences suggest that perturbations in the gut microbiota are associated with graft-versus-host disease (GVHD), and dominance of Enterobacteriaceae is related to poor prognosis after allogeneic hematopoietic stem cell transplantation (SCT) (Taur Y, Blood. 2014; 124:1174-1182). We recently reported that degree of goblet-cell loss was significantly corelated with poor prognosis in 90 patients who underwent SCT in our institute (Ara, et al. 2018 Tandem BMT meeting #220). Goblet cells play a critical role in forming the mucus layer that constitutes not only physical but also chemical barrier, by retaining antimicrobial peptides, against invading microbes from gut lumen. In the current study, we explored the mechanism by which goblet cells protect recipients against GVHD, especially focusing on the role of antimicrobial peptide Lypd8 that is produced by colon epithelial cells and specifically suppresses motilities and biotranslocation of flagellated bacteria including harmful Enterobacteriaceae. [Methods] Mice were lethally irradiated and injected with 5 × 106 bone marrow cells and 7.5 × 106 splenocytes from allogeneic or syngeneic donors on day 0. Recipient mice were intraperitoneally injected with 0.3 mg recombinant mouse IL-25 (rmIL-25) or vehicle from day -6 to 0. [Results] In the B6 → B6D2F1 model, goblet cells in the colon were significantly decreased in association with severe GVHD (Figure A). Fluorescent in situ hybridization (FISH) using the universal bacterial probe EUB338 showed bacterial translocation to the colonic mucosa after SCT. Quantitative PCR targeting bacterial 16S rRNA confirmed that bacterial load in the lamina propria was significantly increased in allogeneic mice compared to syngeneic controls and naive mice (Figure B). Immunofluorescent staining showed Lypd8 at the border of the inner mucus layer and colonic epithelial cells was reduced in allogeneic mice. To evaluate role of Lypd8 in GVHD, lethally irradiated B6-Lypd8-/- mice and wild type (WT) B6 controls were transplanted from BALB/c mice. Both FISH and quantitative PCR showed an increased bacterial translocation into the colonic mucosa in Lypd8-deficient recipients compared to WT recipients (Figure C), indicating that Lypd8 plays a protective role against bacterial translocation into the colonic mucosa. In association with enhanced bacterial translocation, significantly more donor T cells were infiltrated into the gut and liver in Lypd8-/- recipients compared to WT controls (Figure D). Strikingly, GVHD was significantly more severe with shorter survival in B6-Lypd8-/- mice compared to WT recipients (Figure E). GVHD exacerbation in Lypd8-/- mice was reproduced when Lypd8-/- recipients were co-housed with WT recipients for 4 weeks before SCT, excluding the virulent microbiota in Lypd8-/- mice as the potential mechanism of GVHD exacerbation in these mice. Finally, we tested if the goblet-cell protection could attenuate GVHD. Pre-transplant administration of IL-25 mitigated goblet-cell loss and bacterial translocation, reduced plasma levels of IFN-g and IL-6, and ameliorated GVHD mortality. Protective effects of pre-transplant IL-25 was abrogated when Lypd8-/- mice were used as recipients, suggesting that goblet cells protect recipients against GVHD via a Lypd8-dependent manner. [Conclusion] Our results demonstrated that goblet cells suppress bacterial translocation into the colon mucosa and play a protective role against GVHD via a Lypd8-dependent manner. Since increase in flagellated bacteria in the gut could be associated with GVHD exacerbation, goblet cells and Lypd8 could be potentially prophylactic and therapeutic targets for GVHD. Figures: (A) The amount of goblet cells in the colon of allogeneic or syngeneic recipients, or naïve mice are shown. (B) Colon samples were harvested from syngeneic and allogeneic recipients on day +7 after SCT. DNA was extracted from colon samples after removing epithelial cells by incubating with EDTA and subjected to quantitative PCR with 16S rRNA specific primers. (C-E) WT or Lypd8-/- B6 mice were lethally irradiated and transplanted from allogeneic BALB/c mice. Bacterial load in the colon lamina propria (C) and absolute numbers of donor T cells in the liver and colon (D) on day +5, and survival curves (E) after SCT are shown. *; p<0.05, **; p<0.01, ***; p<0.005. Disclosures No relevant conflicts of interest to declare.

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Foxo1 controls gut homeostasis and commensalism by regulating mucus secretion

Journal of Experimental Medicine ◽

10.1084/jem.20210324 ◽

2021 ◽

Vol 218 (9) ◽

Author(s):

Zuojia Chen ◽

Jialie Luo ◽

Jian Li ◽

Girak Kim ◽

Eric S. Chen ◽

...

Keyword(s):

Goblet Cell ◽

Cell Function ◽

Intestinal Inflammation ◽

Intestinal Barrier ◽

Goblet Cells ◽

Short Chain Fatty Acids ◽

Mucus Secretion ◽

Intestinal Epithelial ◽

Forkhead Box ◽

Junction Proteins

Mucus produced by goblet cells in the gastrointestinal tract forms a biological barrier that protects the intestine from invasion by commensals and pathogens. However, the host-derived regulatory network that controls mucus secretion and thereby changes gut microbiota has not been well studied. Here, we identify that Forkhead box protein O1 (Foxo1) regulates mucus secretion by goblet cells and determines intestinal homeostasis. Loss of Foxo1 in intestinal epithelial cells (IECs) results in defects in goblet cell autophagy and mucus secretion, leading to an impaired gut microenvironment and dysbiosis. Subsequently, due to changes in microbiota and disruption in microbiome metabolites of short-chain fatty acids, Foxo1 deficiency results in altered organization of tight junction proteins and enhanced susceptibility to intestinal inflammation. Our study demonstrates that Foxo1 is crucial for IECs to establish commensalism and maintain intestinal barrier integrity by regulating goblet cell function.

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